This application contains the following objectives: (1) characterization of squid nerve DFPase; (2) search for the natural substrate and physiological role for DFPase; (3) examination of direct action of organophosphorus (OP) cholinesterase (ChE) inhibitors; (4) study of the spontaneous reactivation of irreversibly inhibited ChE. DFPase will be purified by electrofocussing. Enzyme will be characterized by using DFP and Tabun, and by noting the effects of Mn ions. Enzymatic hydrolysis of DFP, Tabun, and Soman will be compared in CD spectropolarimeter for stereospecificity of DFPase and conformational changes in the enzyme. A parallel between DFPase and isethionate, and a role for DFPase in the metabolic pathway, cysteine to isethionate, will be sought. Penetration of cysteine, cysteic acid, taurine, and sulfinic acid analogues into squid axons and the metabolic steps from one to another of these intermediates will be examined. Accumulation of intermediates in the presence of DFPase inhibitors, or excess, DFP, will be determined. Intermediates will be sought which qualify as natural substrates for DFPase, e.g., cysteine sulfinic acid or hypotaurine coupled through the -SO2H to ATP, CoA, etc. Direct effects of OP compounds on excitable membranes will be explored. Electrophorus cells will be exposed to Soman until electrical activity is blocked, affording an inhibition of ChE which is not reversible by pyridine-2-aldoxime methiodide (PAM). Restoration of electrical function by PAM without restoration of ChE will indicate that block of electrical activity has been due to interactions of the OP with components of the excitable membrane other than ChE. Voltage clamp will be used to explore these effects. Spontaneous reactivation of ChE in OP treated intact cells will be compared to reactivation in membrane preparations. Results will indicate whether reactivation is due to a property of the membrane, to an enzyme produced by the cell which may cleave the P-O serine bond, or to a naturally occurring nucleophilic agent which serves as reactivator. Proposed research represents the application of basic biochemical concepts to fundamental problems of nerve metabolism and functions, and to problems of public concern such as the detoxication of nerve gases and pesticides.